Total chemical synthesis of turkey ovomucoid third domain and an HLE-specific inhibitor

Nicholas William Warne, Purdue University

Abstract

The object of this thesis is in three parts. First, explore the feasibility of chemical synthesis of Avian Ovomucoid Third Domains, a family of Kazal type serine proteinase inhibitors. Second, if the project is worthwhile, develop a system by which OM3 can be generated by total synthesis. Finally, demonstrate that the system works by synthesizing control and test proteins and characterizing them extensively. A 51 residue Turkey ovomucoid third domain (SYN$\sb{(6-56)}$OMTKY3) was synthesized using the RaMPS system from DuPont utilizing FMOC amino terminal protection, t-butyl based side chain protection, HOBt mediated couplings and ACM groups to protect the cysteines. The resultant protein was cleaved from the resin and folded via a mixed disulfide with glutathione intermediate. Once folded, the protein was purified and characterized both functionally and structurally. The association equilibrium constants for the synthetic inhibitor and six serine proteinases were determined and found to be identical to those of the natural inhibitor (OMTKY3). The synthetic protein was identical to a 51 residue semi-synthetic inhibitor when compared chromatographically. Also, the thermal stability of SYN$\sb{(6-56)}$OMTKY3 was determined and found to be identical to the semi-synthetic sample. PDMS and Ion-Spray MS of SYN$\sb{(6-56)}$OMTKY3 showed that it had the expected mass. This protein was thus found to be identical to standards in our lab. A designed inhibitor was synthesized that was specific for Human Leukocyte Elastase and was found to be very specific when compared against five different enzymes, however, the inherent fluorescence of the molecule was different from what was expected, indicating that the protein may be misfolded. The thermal stability of the HLE specific inhibitor was examined by CD and found to be greater than that for SYN$\sb{(6-56)}$OMTKY3, indicating that the protein is properly folded, but not explaining the problem with fluorescence. This problem was examined and found to not be due to misfolding of the protein but rather to difficulties of fluorescence. Recombinant techniques were used to generate a protein similar to OMTKY3 and it was found that this approach was also successful. The development of these techniques for generating variants of OM3 will aid in our future understanding of how the sequence of OM3 affects its function.

Degree

Ph.D.

Advisors

Laskowski, Purdue University.

Subject Area

Biochemistry

COinS